SEANET Research Theme: Aquaculture in a changing ecosystem

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1 Date Submitted: September 14 th, 2017 Student Name: Michele Condon Year of School: Junior Department of Marine Sciences Faculty Mentor: Dr. Carrie Byron, Adam St. Gelais SEANET Research Theme: Aquaculture in a changing ecosystem Project Title: A Histological Condition Assessment of Farmed Blue Mussels (Mytilus edulis) in Casco Bay, Maine Using Reproductive and Storage Tissue Analysis Dates covered by this report: Feb-Aug 2017

2 Abstract: The motivation for this work was to help blue mussel (Mytilus edulis) farmers better understand the health of the mussels they are farming with. Histological slides were produced and analyzed through qualitative and quantitative measures. Gonads were staged to help determine the reproductive development of the blue mussel population as a whole. The blue mussels were fully developed in late May and had undergone spawning by June. The fraction of gonad and storage tissue (vesicular connective tissue and adipogranular tissue) found in the mantle was calculated using Image- J. Gonad tissue was at is highest percentage within the mantle during July, which resulted in a lower fraction of storage tissue. Key words: Mytilus edulis - histology - reproduction - storage tissue - aquaculture Introduction: Shellfish aquaculture is a growing industry as it is able to produce food, jobs and also environmental benefits. Shellfish, especially blue mussels, are known as water purifiers and environmentally sensitive indicators (Shumway 2003). Bangs Island Mussels, located in Casco Bay, Maine is a family-owned sustainable mussel and kelp farm. In the summer of 2016, the farm experienced a mass blue mussel mortality event that resulted in a large financial loss. There is interest in investigating why this happened in order to prevent future losses and improve current farming practices. Understanding the health of the blue mussel in regards to reproductive and storage tissue is one method in doing so. Providing as much information as possible to the farmers can help them make adjustments to their farms to let them thrive. The major research question that was asked was how reproduction and storage tissue fractions of blue mussels varied seasonally. Qualitative and quantitative measurements will be taken of reproductive and storage tissue structures by the means of histology (the use of staining techniques to look at the tissue level of an organism via microscopy) and image analysis. There has been limited research published on the use of histology to analyze the reproductive and storage tissues of blue mussels, but none conducted in the Gulf of Maine. My proposed research will expand on this prior histology research of using reproductive and storage tissues structures as a form of analysis on the health of blue mussels by collecting blue mussels from Casco Bay, Maine. Duinker et al. (2008) used histology to look at reproductive stages of blue mussels over time in Norway. A classification system was made of the gonad developmental stages modified from Chipperfield (1953). A similar classification system of storage tissue stages was made for this study. When adipogranular tissue (lipid and protein storage) and vesicular connective tissue (glycogen storage) are classified, it can be a helpful tool in the assessment of energy investment. This research relates to SEANET, as it will provide helpful information to blue mussel farms. The research of blue mussels is directly related to sustainable ecological aquaculture. Aquaculture is important on Maine s coast, as it is many people s livelihood.

Methods: Five mussels were collected from each of Bangs Islands Mussel farm site during monthly sampling trips.

The mussels were rinsed and stored in 70% ethanol until the anterior portion of the mussel could be embedded in paraffin.

The produced slides were analyzed under a light microscope. Each mussel was first identified as male or female based on the gonads present.

3 Methods: Five mussels were collected from each of Bangs Islands Mussel farm site during monthly sampling trips. Shell length, width, height, and weights were recorded before the mussels were shucked out of the shell to be fixed in a buffered 10% formalin solution for 24 hours. The mussels were rinsed and stored in 70% ethanol until the anterior portion of the mussel could be embedded in paraffin. These paraffin blocks were sectioned (5 sections at 5 um thickness from the mussel moving at 1000 um increments distally) and stained with Hematoxylin and Eosin. The produced slides were analyzed under a light microscope. Each mussel was first identified as male or female based on the gonads present. The screen was filled with healthy gonads (100x) and a picture was saved. The first 10 gonads observed were staged using a modified classification (Duinker et al 2008). Female gonad development Stage 0 Little gonad tissue Can not determine sex Male gonad development Stage 0- Little gonad tissue Can not determine sex Stage 1 Sex determined due to oocytes present Start of egg yolk formation shown with light purple circles forming around acini Stage 1- Sex determined due to sperm cells present Immature sperm cells make up most, if not all of acini radius

Stage 2- Most oocytes have egg yolks Most oocytes still

Stage 2- Immature sperm cells still abundant (at least ¼ of

present Stage 3- All oocytes have egg yolks Higher proportion

tissue Stage 3 Higher proportion of mature gametes Less than 10

4 Stage 2- Most oocytes have egg yolks Most oocytes still attached to acini wall but some may be detached and mature Stage 2- Immature sperm cells still abundant (at least ¼ of acini radius, usually 10 dark cells on acini wall) Spermatids present Stage 3- All oocytes have egg yolks Higher proportion of mature eggs in acini Higher quantity of gonad area in mantle tissue Stage 3 Higher proportion of mature gametes Less than 10 darker cells on the acini wall Mature sperm cells outweigh immature sperm cell

This process was continued on another section of the mantle tissue on the same slide. This was then repeated on another slide of the same mussel. The pictures taken were then analyzed through Image-J.

5 This process was continued on another section of the mantle tissue on the same slide. This was then repeated on another slide of the same mussel. The pictures taken were then analyzed through Image-J. The total amount of pixels of gametes, adipogranular tissue, and vesicular connective tissue were found in proportion to the total amount of pixels that made up the mantle tissue. A color threshold was used to determine the gametes, adipogranular tissue, and vesicular connective tissue (Figure 1). Figure 1. Image-J analysis using color threshold. The gonad area is highlighted based on color and the pixels were counted. Results: For female blue mussels the highest abundance of developed gonads occurred in late May as 93 % of the gonads were staged at a 3 (Figure 2). For male blue mussels, the highest abundance of developed gonads occurred in late May and June. 69% of the gonads were stage at 3 in late may while 69.6% were staged at 3 in June (Figure 3). This increase in stage 3 gonads decreased the presence of the other stages. For female blue mussels in late May, there was 0% at stage 1 with 7% at stage 2 (Figure 2). For male blue mussels in late May, there was 8.3% at stage 1 and 22.8 % at stage 2. For male blue mussels in June, there was 9.1% at stage 1 and 21.3% at stage 2 (Figure 3). For female blue mussels, there was only a presence of stage 0 mussels in February (1.5%) and August (14.5%) (Figure 2). For male blue mussels, there was only a presence of stage 0 in August (6.7%)(Figure 3). For female blue mussels, the average gonad fraction reaches its peak in April at 55.3% of the mantle tissue. The average vesicular connective tissue fraction is highest in February (36.5%) and lowest in June (6.3%). The average adipogranular tissue fraction is lowest in May (2.9%). Unaccounted for structures in the mantle tissue were highest in June (39%) and August (48.4%) (Figure 4). For male blue mussels, the average gonad fraction reached a peak in March (74.7%) and steadily decreased. The average vesicular connective tissue fraction and adipogranular connective tissue was highest in February.

6 Percent Percent Unaccounted for structures in the mantle tissue were highest in August (29.3%) (Figure 5) Reproductive Stages of Female Mussels /2/17 3/23/17 5/1/17 5/31/17 6/14/17 7/6/17 8/8/17 Date Stage 3 Stage 2 Stage 1 Stage 0 Figure 2. Total percent of gonads at varying developmental stages in female blue mussels collected from Bangs Island and Clapboard Island. 100 Reproductive Stages of Male Mussels Stage 3 Stage 2 Stage 1 Stage 0 0 2/2/17 3/23/17 5/1/17 5/31/17 6/14/17 7/6/17 8/8/17 Date Figure 3. Total percent of gonads at varying developmental stages in male blue mussels collected from Bangs Island and Clapboard Island.

7 Percent Percent Fractions of Mantle Tissue of Female Mussels Average Gonad Fraction Average Vesicular Connective Tissue Fraction Average Adiopoganular Tissue Fraction Average Other Fraction 0 FEB MAR APR MAY JUN JUL AUG Month Figure 4. Fractions of gonad area, vesicular connective tissue, and adipogranular tissue present in the mantle tissue of female blue mussels. Fractions of Mantle Tissue of Male Mussels Average Gonad Fraction Average Vesicular Connective Tissue Fraction Average Adiopogranular Tissue Fraction Average Other Fraction 0 FEB MAR APR MAY JUN JUL AUG Month Figure 5. Fractions of gonad area, vesicular connective tissue, and adipogranular tissue present in the mantle tissue of male blue mussels. Discussion: By staging the gonads, a better understanding can be gained of when the mussels are most developed. This can then help determine when the mussel is ready to spawn.

8 The peak spawning time is important for farmers to be aware of because spawning can result in a large loss of the mantle tissue (Duinker 2008). A loss of mantle tissue is a direct loss in meat yield. The peak spawning time has yet to be determined for blue mussels in the Gulf of Maine due to varying environmental factors (Chipperfield 1953). There is usually a reported spawning in early spring followed by a possible secondary spawning in late summer. A peak spawning of the blue mussels in late May was observed from the data and will now expand the database on peak spawning time to the Gulf of Maine. This peak spawning time was determined based on when there was a high percentage of stage 3 female gamete followed by a sharp decrease. This was seen in late May with 93% stage 3 gametes followed by only 65 % being stage 3 gametes. This suggests that spawning happened sometime between these two dates because there was a loss in fully developed eggs. In order to complete the staging, a guide was needed to have a standard throughout the process and ensure accuracy. Modifications were made from guidelines set in Duinker (2008). There was a guide made for both male and female blue mussels. Multiple characteristics were taken into account such as gonad abundance, size, and features. By determining how much gonad area is in the mantle tissue allows us to see how much energy is being invested into reproduction. When the gonad area is at a higher percentage such as the 74.7% area in males in March, the mussel is consuming a lot of energy to create sperm. With more energy being invested into reproduction, there is less energy available for storage tissues. Less energy being invested in creating adipogranular tissue results in less protein and lipid stores and less energy being invest in creating vesticular connective tissue will result in less glycogen stores. A decrease in these makes the mussel less resilient and more vulnerable to environmental stress, food shortage or diseases (Bolch 2009). Even more energy is needed to make female eggs rather than male sperm. A 55.3% gonad area in females in April would mean a large investment is being made into reproduction. There are connections that can be made from Figure 2 and Figure 4. It was determined from Figure 2 that the peak spawning time between late May and June. At this time, there is the lowest fraction of vesicular connective tissue at 8.3% and a low fraction of adipogranular tissue at 6.3%. During spawning, the female mussels will have the lowest energy reserves as all their energy is consumed by making eggs. These lower levels of energy reserves will make it more difficult for the mussels to combat stressful situations. There was also a large percent of the mantle tissue of other area in the mantle tissue. This is area that was unaccounted for by gonads, vesicular connective tissue, or adipogranular tissue. This could be due to possible open areas in the tissue or inaccurate Image-J calculations errors. In late August, the increase in this percentage was due to hemocytes (blood cells in invertebrates) found. Possible further research will include continuing staging and Image-J analysis through winter months. Weekly samples rather than monthly samples would provide a more accurate timeline for spawning. Staging of storage tissues will give an idea of the development throughout this timeline. Calculating the condition index would be a useful tool to determine the meat yield and then make comparisons to gonad area.

9 Conclusion: A better understanding of reproductive development, spawning time, and energy investment was gained on blue mussels collected from Casco Bay, Maine over the months of February to August. This information is valuable to mussel farmers who want a greater meat yield. More information can be collected on a greater scale to improve the mussel industry even more. References: Bolch C, Moltschaniwskyj N Energy storage and reproduction in mussels, Mytilus galloprovincialis: the influence of diet quality. Journal of Shellfish Research. 28(2): Chipperfield PJ Observations on the breeding and settlement of Mytilus edulis in British waters. Journal of the Marine Biological Association of the UK 32(2): Duinker A, Håland L, Hovgaard P, Mortensen S. (2008). Gonad development and spawning in one and two year old mussels (Mytilus edulis) from Western Norway. Journal of the Marine Biological Association of the UK. 88(07): Shumway S, Davic C, Downey R, Karney R, Kraeuters J, Parsons J, Rheault R, Wikfors G, (2003) Shellfish Aquaculture-in praise of sustainable economies and environments World Aquaculture 34(4): 8-10

Corina CIOCAN National Institute for Marine Research and Development Grigore Antipa Constanta

Cercetari marine I.N.C.D.M. Nr. 34 29-33 2002 LYSOSOMAL MEMBRANE STABILITY IN BLOOD CELLS OF THE SOFT SHELLED CLAM (Mya arenaria L.) Corina CIOCAN National Institute for Marine Research and Development